Molecular and biological characterization of a partitivirus from Paecilomyces variotii

Paeciliomyces variotii is a thermo-tolerant, ubiquitous fungus commonly found in food products, indoor environments, soil and clinical samples. It is a well-known biocontrol agent used against phytopathogenic fungi and its metabolites have many industrial applications. Rare reports of P. variotii-related human infections have been found in the medical literature. In this study, we report for the first time the infection of P. variotii isolated from a soil sample collected in a rice field with a double-stranded RNA virus, Paeciliomyces variotii partitivirus 1 (PvPV-1) in the family Partitiviridae. P. variotii harboured icosahedral virus particles 30 nm in diameter with two dsRNA segments 1758 and 1356 bp long. Both dsRNA1 and dsRNA2 have a single open reading frame encoding proteins of 63 and 40 kDa, respectively. These proteins have significant similarity to the RNA-dependent RNA polymerase and capsid protein encoded by the genomic segments of several viruses from the family Partitiviridae. Phylogenetic analysis revealed that PvPV-1 belongs to the family Partitiviridae but in an unclassified group/genus, tentatively nominated Zetapartitivirus. PvPV-1 was found to increase the growth rate of the host fungus, as indicated by time course experiments performed on a range of different media for virus-infected and virus-free isogenic lines. Further, dual-culture assays performed for both isogenic lines confirmed the antagonistic potential of P. variotii against other phytopathogenic fungi. The findings of this study assist us in understanding P. variotii as a potential biocontrol agent, together with plant–fungus–virus interactions.


InTRoduCTIon
The soil hosts a highly diverse fungal network that is actively involved in the disintegration of organic matter, thus shaping the structures and functions of microbial and plant communities.Soil fungi have roles in organic matter stabilization by producing enzymes fixing nitrogen, controlling root pathogens and protecting against drought [1][2][3][4].The diversity of fungal communities is greatly influenced by plant diversity and reciprocally fungal communities affect plant growth through different associations, including mutualism, antagonism or pathogenicity [5].
Paeciliomyces variotii has a potential role in controlling pests and diseases and this ability makes it an eco-friendly alternative to other conventional agricultural practices.P. variotii has been shown to be an effective antagonist, reducing disease severity against phytopathogenic fungi and bacteria, including Podosphaera xanthii, Fusarium oxysporum, Macrophomina phaseolina, Mycosphaerella melonis and Xanthomonas spp.[18].P. variotii was found to be compatible with fungicides such as cymoxanil, mancozeb, triadimenol, copper hydroxide and copper oxychloride [18].Therefore, P. variotii may be utilized as a biological control agent against several diseases and should be considered for potential integration into advanced pest management strategies.
P. variotii can also produce secondary metabolites, including the antibiotic ascofuranone, peptides, polyketides, naphthopyranones, sphingofungins, novel branched fatty acids, eicosenoic acids, anacardic acid analogues and high-value volatile compounds.Many of these products play a role in improving animal and human health or as herbicidal agents for agrochemical markets [19].
The presence of a mycovirus in P. variotii may result in virus-host interactions influencing P. variotii as a biocontrol agent, by affecting growth and metabolite production.At present, the focus of mycovirology is expanding from plant pathogenic fungi and mycovirus-mediated hypovirulence to other interactions; effects such as hypervirulence, metabolite production, drug resistance, control of endophytic traits and other mycovirus-facilitated phenotypes are also central to current research [20].For fungi such as P. variotii, which act as natural biocontrol agents for other phytopathogenic fungi [18], investigations of virulence within this three-way interaction are paramount.This investigation is the first to report the presence and genomic sequence of a partitivirus in P. variotii and assess the effects of mycovirus infection on the biology of the host fungus.

METHodS
Isolation and identification of P. variotii P. variotii was isolated from soil samples collected in rice fields from different regions of Khyber Pakhtunkhwa, Pakistan.Soil samples were serially diluted, inoculated on potato dextrose agar (PDA, Millipore) and incubated at 25 °C for 5-7 days followed by single colony isolation on PDA.Total nucleic acid and dsRNA extractions were performed [21,22], and fungal mycelia were stored in 40 % glycerol at −20 °C for future use.For molecular identification of fungi, the internal transcribed spacer (ITS) regions of nuclear DNA were amplified by polymerase chain reaction (PCR) using ITS1 and ITS4 primers [23,24].

Fragmented and primer-ligated dsRnA sequencing (FLdS)
Purified dsRNA was subjected to a novel method of library construction called FLDS [22].This method consists of physical fragmentation of dsRNA following cellulose column chromatography, synthesis of complementary (c) DNA by reverse transcription (RT) using a modified rapid amplification of cDNA ends (RACE) method, and library construction and amplification of cDNA via PCR.Finally, dsRNA sequencing was performed using the KAPA Hyper Prep kit Illumina Platform (Kapa Biosystems, Woburn MA, USA).Illumina NovaSeq 6000/PE150 was used to determine 150 bp of the paired end sequence of each fragment.Raw sequencing data were processed through the FLDS pipeline (available in GitHub).CLC Genomic Workbench version 11.0 was used for de novo assembly of contigs >300 nt (other options were default).Full-length recovery of dsRNA segments was confirmed by manual analysis using CLC Genomic Workbench version 11.0, Genetyx version 14 and Tablet viewer version 1.19.09.03.

Multiple sequence alignment, phylogenetic analysis and secondary structure prediction
The online Multiple Alignment using Fast Fourier Transform (MAFFT v7.511) tool was used for multiple sequence analysis [25].Molecular Evolutionary Genetics Analysis (MEGA) 11 software was used for phylogenetic analysis [26].The secondary structures of the 5′-and 3′-termini of dsRNAs were determined using the online RNAfold (2.5.1) tool [27].

Purification of virus-like particles (VLPs)
VLPs were purified as described elsewhere [28] with some modifications.Fungal mycelium (50 g) was homogenized for ~3 min in two volumes of TE buffer (50 mM Tris-Cl pH 7.5, 1 mM EDTA pH 8.0) in a blender.The homogenate was filtered through Miracloth and put into 250 ml Nalgene bottles.Centrifugation was performed at 10 000 g for 20 min.The VLPs in the supernatant were then precipitated with 10 % (w/v) polyethylene glycol (PEG)-6000 and 0.6 M NaCl by stirring at 4 °C overnight.Centrifugation for 20 min at 10 000 g was performed, and the pellet containing the VLPs was resuspended in TE buffer and recentrifuged at 10000 g for 20 min.The collected supernatant containing the VLPs was then subjected to ultracentrifugation at 105 000 g for 90 min.The pellet containing the VLPs was resuspended in 1 ml TE buffer and transferred to 1.5 ml Eppendorf tubes.The tubes were again centrifuged at 10 000 g for 20 min to further clarify the supernatant containing the VLPs.Virus dsRNA was extracted with phenol-chloroform-isoamyl alcohol, electrophoresed on 1 % (w/v) agarose gel and visualized under ultraviolet light.

Transmission electron microscopy (TEM) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SdS-PAGE)
Purified VLPs were visualized using TEM.Carbon Film 300 Mesh copper grids were glow discharged for 1 min under vacuum and 3 µl of sample was added to the surface of the grids and incubated for 1 min.Excess sample was wiped from the grid manually.Uranyl acetate (2 %) staining solution was added to the surface of the grid and incubated for 1 min.Excess stain was again blotted off the grid manually and the grids were observed by TEM operating at 120 kV using a LAB6 filament.Emission was set to 4 μA, and images were recorded at magnifications ranging from 46 000-67 000×, with an exposure time of 1.4 s.SDS-PAGE was performed for the purified VLPs [29], followed by SYPRO-RUBY (Thermo Fisher Scientific) staining.

Curing of mycovirus infection with cycloheximide treatment
Fungal isolates were cured of mycovirus infection by treatment with cycloheximide [30][31][32], which was filter-sterilized and added into PDA.Growth was restricted by high concentrations of cycloheximide, therefore the culture was selected and placed into a 2 ml Eppendorf tube containing water.Following shaking and dilution, the contents of the tube were inoculated on water agar plates.Individual spores were selected and inoculated onto freshly prepared PDA plates and subsequently potato dextrose broth (PDB) for nucleic acid extraction.Virus-free isogenic lines were confirmed by performing RT-PCR amplification using a standard protocol with purified dsRNA (9 µl) as template, which was heat-denatured with 100 % dimethyl sulfoxide (DMSO; 40 µl) and random hexamers (1 µl) at 65 °C for 20 min.The denatured dsRNA was snap-cooled on ice and precipitated using 0.1 vol of 3 M sodium acetate (0.5 µl) pH 5.2 and 2.5 volumes of absolute ethanol.The mixture was incubated at −80 °C for 30 min or −20 °C overnight followed by centrifugation at 21 380 g for 20 min.The pellet was washed with 70 % ethanol, air-dried, incubated for 10 min on ice and resuspended in a first-strand cDNA synthesis reaction [4 µl nuclease-free water, 4 µl RT buffer (5×), 5 µl deoxyribonucleotides (dNTPs; 2 mM), 2 µl dithiothreitol (DTT; 0.1 M), 3 µl random hexamer primers (10 pmol)], ribonuclease inhibitor (Thermo Fisher Scientific) and RTase (SuperScript, Thermo Scientific).Next, the reaction mixture was incubated at 37 °C for 90 min.The cDNA was diluted 1 : 4 (10 µl of cDNA and 40 µl of sterile distilled water) and PCR was performed using a virus-specific reverse primer (5′-GAT AGA GCG CCA CGC ACA CCG-3′) and forward primer (5′-CCG TAC AAA CGA GGA GTG TTG-3′), designed from the virus sequences obtained through FLDS to amplify a specific fragment of the virus genome.The provenance of the RT-PCR amplicon was confirmed by restriction endonuclease digestion with EcoRV (Promega) according to the manufacturer's instructions.

Horizontal transfer of the mycovirus via hyphal fusion
The mycovirus was transferred from the original virus-infected isolate (donor) to one cured of infection with cycloheximide treatment (recipient).The two isolates were allowed to grow and fuse on PDA, followed by selection of different regions from both donor and recipient, and sub-culturing on fresh PDA plates.The cultures were subsequently inoculated in PDB, and their infection status was determined by dsRNA extraction and agarose gel electrophoresis.

Radial expansion, biomass production and metabolic assays
For radial expansion assays and assessment of colony morphology, equal numbers of spores of virus-infected and virus-free isogenic lines were centrally inoculated on PDA and incubated at 28 °C.Other media investigated included Sabouraud dextrose agar (SDA), yeast extract and sucrose agar (YES) and Czapek Dox agar (CDA) complete and minimal media.Colony radii were measured and recorded every 24 h over a period of 4 days until the colonies completely covered the plates.
For biomass production assays, equal numbers of spores of virus-infected and virus-free isogenic lines were inoculated into 10 ml PDB in 50 ml Falcon tubes, incubated at 28 °C and further processed once visible mycelial mats had grown.Individual cultures were filtered through Whatman paper, wet mycelial weights were recorded, mycelia were wrapped in filter paper and dried for 24 h, and dry mycelial weights were recorded.
2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)−5-carboxanilide-2H-tetrazolium (XTT) metabolic assays were performed as described elsewhere [33,34].Fungi were grown on PDA until sporulation occurred and conidia were collected in 1× phosphate-buffered saline (PBS) and counted using a haemocytometer.Fungal conidia (10 7 spores) were seeded in malt extract broth (MEB), YES and different formulations of CD liquid minimal media in 96-well plates and allowed to grow at 25 °C for 24 h.XTT-menadione-liquid media solution was added to each well and the 96-well plates were incubated in the dark for 1 h.The supernatants from each well were transferred to a fresh 96-well plate and assayed using a plate reader at 490 nm.All experiments were performed in triplicate and the results were analysed statistically using the two-tailed Student's t-test or two-way analysis of variance (ANOVA) in GraphPad Prism.

dual-culture assays
The antagonistic potential of P. variotii virus-infected and virus-free isogenic lines was evaluated against other fungi, including Aspergillus terreus, Hypoxylon spp.and Penicillium citrinum by performing dual-culture experiments [35,36].These fungal isolates were isolated from tobacco field soil samples and tested negative for mycovirus infection following dsRNA extraction.Equal numbers of spores of both P. variotii and the antagonist were inoculated on freshly prepared PDA media, 2 cm away from the edge of the PDA plates.In the control group, PDA plates were inoculated solely with the antagonist in the absence of P. variotii.Plates were kept in an incubator at 25 °C for 5-7 days until inhibition could be observed.All experiments were performed in octuplicate and the following formula was used to calculate % inhibition.

% inhibition = (A−B)/A×100
A=colony diameter of fungal pathogen in the control group B=colony diameter of fungal pathogen in the treated group

RESuLTS
Identification of a dsRnA mycovirus in a soil isolate of P. variotii A total of 8 rice field soil samples were screened, resulting in 46 fungal isolate representatives of genera such as Aspergillus, Fusarium, Hypoxylon and Trichoderma.Following screening for mycovirus infection, five tested positive for mycoviral infection, including a P. variotii isolate found to be infected with dsRNA elements, potentially representing the genome or the replicative form of an RNA mycovirus.One fungal isolate, 15R, was found to harbour such dsRNA elements between 1 and 2 kbp in size.The colony morphology of the virus-infected P. variotii 15R isolate and its dsRNA electrophoretic profile are shown in Fig. 1a,  c, respectively.Minor bands observed in Fig. 1c are potentially ribosomal (r) RNAs from the fungal host, since no S1 nuclease treatment was performed following cellulose column chromatography.FLDS was performed using this extract, and only one virus whose genome corresponds to the two clear dsRNA bands was found in the P. variotii 15R isolate.Following purification of VLPs and visualization by TEM, icosahedral VLPs 30 nm in diameter (Fig. 1b) were observed.The proteinaceous component of the VLPs was subjected to SDS-PAGE and staining with SYPRO-RUBY, revealing a single band approximately 40 kDa in size (Fig. 1d).

Molecular characterization of a novel partitivirus infecting P. variotii
The purified dsRNA elements were subjected to FLDS for complete sequence determination.Two contigs containing 57 and 37 % of assembled reads were determined as major (>2.5 % of assembled reads) contigs.Based on the mapping analysis, these two contigs were confirmed to be complete sequences of two dsRNAs, 1758 and 1356 bp in size.The complete nucleotide sequences of dsRNA 1 and dsRNA 2 were submitted to DNA Data Bank of Japan (DDBJ) with accession numbers LC764452 and LC764453, respectively.
The 5′-untranslated regions (UTRs) of PvPV-1 dsRNA1 and dsRNA2 are 14 and 113 bp long, respectively, and show conserved nucleotides at the 5′-termini (AAACTTTTGT) (Fig. 2b).A similar conserved region (AAACTTTTGT) is present in the 5′ UTR of Botryosphaeria dothidea virus 1.The 3′-UTRs differ in length, 94 bp for dsRNA1 and 130 bp for dsRNA2, and show a conserved nucleotide stretch of small and long sequences with a conserved stretch (ATTCCCTCGATTC) at the 3′-terminus (Fig. 2b).This is in accordance with the observations for multi-component RNA viruses, where the 5′-terminal sequences are important for recognition by the RdRP during the replication of viral genome [39].Possible secondary structures were predicted for the 5′-and 3′-termini of both PvPV-1 dsRNA 1 and dsRNA 2 (Fig. S1, available in the online version of this article).Such structures give stability and strength to the RNA molecule or genome and may have a role in viral replication and assembly [40].

Construction of isogenic lines and completion of Koch's postulates
Cycloheximide at 150 mM concentration successfully cured P. variotii from mycoviral infection.The virus-free isogenic line was confirmed by RT-PCR amplification using virus-specific primers.The virus-specific amplicon fractionated by agarose gel electrophoresis was present in the wild-type PvPV-1-infected isolate but absent from the virus-free isogenic line (Fig. 4a).The RT-PCR product was further treated with restriction endonuclease EcoRV to confirm the presence of a virus-specific amplicon, generating, as expected, two fragments that were 319 and 147 bp in size (Fig. 4a, b).Colony morphologies of virus-infected and virus-free isogenic lines on PDA are shown in Fig. 4c.
Subsequently, hyphal fusion between virus-infected (donor) and virus-free (recipient) isogenic lines was successfully performed, as shown in Fig. S2a,b, for horizontal transmission of PvPV-1.Selected regions (Fig. S2c) of both donor and recipient mycelia were assessed for mycovirus presence and dsRNA extraction followed by agarose gel electrophoresis confirmed virus transfer from donor to recipient (Fig. S2d).This experiment completes Koch's postulates, an important step for establishing a cause-effect relationship, and ensures genetically identical isogenic lines, as treatment with chemicals such as cycloheximide is known to cause mutations [41].

Effects of PvPV-1 infection on P. variotii radial growth, biomass, metabolism and antagonistic potential
In order to determine the effects of PvPV-1 infection on P. variotii, virus-free and virus-infected isogenic lines were analysed in terms of radial growth on different solid media and biomass production in PDB [42,43].These experiments included the original virus-infected (VI) isolate, the cured virus-free (VF) isolate, and one of the recipients of PvPV-1 following hyphal fusion (R5).
The radial expansion of VI, VF and R5 was measured over a period of up to 5 days on different growth media.In all cases, the radial growth of VI and R5 is faster as compared to VF and this observation was confirmed as statistically significant for some time points using two-way ANOVA (P-value <0.05).The colony morphologies of VI and VF grown on different media are shown together with the graphical representation of the diameters recorded in Fig. 5a, b respectively.Comparisons of VF and R5 radial expansion were performed in smaller Petri dishes and a graphical representation of the diameters recorded is shown in Fig. S2.Further, both the wet and the dry biomass production of VI and R5 were significantly greater (P-value <0.01) as compared to VF, as shown by Student's t-test (Fig. 6a).
Conversely, no statistically significant differences in the metabolic activity of VI and VF P. variotii were noted in MEB and YES (Fig. 6b).However, cultures of VI P. variotii grown in CD minimal media containing sucrose and sodium nitrate as carbon and nitrogen carbon sources, respectively, were darkly pigmented with formazan (a coloured compound produced because of reduction of XTT by the dehydrogenase enzyme produced by metabolically active cells) [44], whereas VF cultures and those deprived of carbon and nitrogen were lighter in colour with minimum optical density values being recorded by spectrometry, presumably because of limited fungal growth (Fig. 6c).The difference between the metabolic activity of VI and VF P. variotii was statistically significant (P-value <0.01), as shown using two-way ANOVA.
Finally, the antagonistic potential of isogenic lines of P. variotii against different fungi, including A. terreus, Hypoxylon spp.and P. citrinum, was evaluated using a dual-culture assay.The percentage inhibition, ranging from 30 to over 50%, was found to be greater for the VI line as compared to the VF line (Fig. 7a, b).This overall trend was shown to be statistically significant (P-value <0.01) when comparing the effect of isogenic lines using two-way ANOVA, although pairwise comparisons did not reveal any significance.

dISCuSSIon
P. variotii is a cosmopolitan fungus that has a significant role as a biocontrol agent and in industry as a source of industrial tannase [18,45].To date, there have been no reports of mycovirus infection in P. variotii and this study is the first to describe the presence of icosahedral VLPs and dsRNA elements from a novel mycovirus, PvPV-1.PvPV-1 has been unambiguously identified as a new member of the family Partitiviridae and has all the traditional hallmarks of a partitivirus: non-enveloped icosahedral VLPs [46] that encapsidate two genomic dsRNA segments 1.3-2.5 kbp in size encoding respectively RdRP and CP and show high sequence identity of their 5′-UTRs [39].The presence of one or more additional genomic components has been reported in several partitiviruses, but there is no such evidence for PvPV-1.PvPV-1 RdRP and CP are both significantly similar in sequence to proteins encoded by Aspergillus flavus partitivirus 1, Aspergillus niger partitivirus 1, Botryosphaeria dothidea virus 1, Colletotrichum acutatum RNA virus 1 and Valsa cypri partitivirus (Table S1).Phylogenetic analysis indicates that this group of mycoviruses clusters in an unclassified taxon unrelated to the existing five genera within Partitiviridae (Fig. 3).Based on our findings, PvPV-1, together with related viruses, represents the new genus named Zetapartitivirus [8] according to the Partitiviridae nomenclature.The species demarcation criteria within each genus of the family Partitiviridae are ≤90 and ≤80 % aa sequence identity in respectively the RdRP and the CP; therefore, PvPV-1 represents a new species within Zetapartitivirus.
Investigating mycovirus infection in fungi used as biocontrol agents is important, as their antagonistic potential may vary, depending on mycovirus-mediated phenotypes, including effects on host growth.For instance, mycovirus-mediated increased host growth may be advantageous, since it could restrict growth of other phytopathogenic fungi, reducing pathogenicity and controlling disease.Growth inhibition of pathogenic fungi by a biocontrol agent, such as mycovirus-infected Trichoderma spp., was previously assessed to investigate potential differences in percentage inhibition.Trichoderma harzianum partitivirus 1 (ThPV-1) infection led to no significant differences in colony morphology or pigmentation of the host Trichoderma harzianum.However, growth inhibition of Pleurotus ostreatus and Rhizoctonia solani by T. harzianum was increased in the VI strain as compared with the VF isogenic strain in dual-culture experiments.Moreover, β−1,3-glucanase but not chitinase activity was significantly increased in the VI strain, suggesting that ThPV-1 has a role in regulating the activity of a specific host enzyme [51].A study on two Trichoderma spp.strains infected with other mycoviruses beyond Partitiviridae reported that mycovirus infection decreased their growth rate, sporulation and biocontrol efficacy [60].Finally, dsRNA elements in T. harzianum were reported to decrease its biocontrol potential but improved plant growth [61].
In this study, PvPV-1 infection increased P. variotii growth based on comparisons of VF and VI isogenic lines in radial expansion assays and biomass measurements in liquid media.This observation is at least partially explained through the enhanced metabolic activity of VI P. variotii, as illustrated by XTT metabolic assays.Additionally, PvPV-1 improved the antagonistic potential of P. variotii by increasing its percentage growth inhibition of pathogenic fungi.This observation may stem from increased growth of the VI isolate as compared to the VF isolate.Our results are in accordance with previous studies illustrating the antagonistic potential of P. variotii [18,62,63].In conclusion, we isolated and characterized a novel partitivirus that confers interesting phenotypes on its host and to our knowledge this represents the first report of mycovirus infection in P. variotii.

Fig. 1 .
Fig. 1.PvPV-1 components.(a) Colony morphology of P. variotii isolate 15R on PDA.(b) TEM visualization of icosahedral VLPs 30 nm in diameter extracted from P. variotii.(c) Electrophoretic profile of the dsRNA elements extracted from P. variotii on a 1 % (w/v) agarose gel, showing two dsRNA bands approximately 1.5 and 1.9 kbp in size.The molecular sizes of λ-EcoT141digested DNA markers are indicated on the left of the gel.(d) Electrophoretic profile of the VLPs on 10 % (w/v) SDS-PAGE analysis of the purified virus particles showing a CP approximately 40 kDa in mass.The molecular sizes of PAGE Ruler unstained protein ladders are indicated on the left of the gel.

Fig. 2 .
Fig. 2. PvPV-1 genomic organization.(a) Schematic representation of PvPV-1 dsRNA 1 and dsRNA 2. Each dsRNA is shown as a black line and each ORF as a grey box.(b) Nucleotide sequence alignment of the 5′-and 3′-termini of the coding strands of the two PvPV-1 dsRNAs using MAFFT.Asterisks signify identical nucleotides.(c) Amino acid sequence alignment of the core RdRP motifs (III-VIII) of PvPV-1 and selected members in the family Partitiviridae using MAFFT.Asterisks signify identical residues; colons signify highly conserved residues and single dots signify less conserved but related residues.

Fig. 3 .
Fig. 3. PvPV-1 phylogeny.Phylogenetic analysis of PvPV-1 and selected members of the family Partitiviridae based on their RdRP amino acid sequences.A multiple alignment of RdRP amino acid sequences was produced using muscle as implemented using mega 11.A neighbour-joining (NJ) phylogenetic tree was constructed using MEGA 11.Bootstrap percentages (1000 replicates) over 50 % are shown.Tips labelled with brown, green and blue shapes indicate that the virus host is respectively fungal, plant or protozoon.The orange rhombus indicates the position of PvPV-1.

Fig. 4 .
Fig. 4. PvPV-1 elimination.(a) Agarose gel electrophoresis of the PvPV-1 RdRP amplicon following RT-PCR of P. variotii virus-infected (VI) and virusfree (VF) isogenic lines before and after digestion with EcoRV.The molecular sizes of the Thermo Fisher Scientific 1 kbp DNA marker are indicated on the left of the gels.(b) The PvPV-1 RdRP amplicon sequence (466 bp) showing the forward and reverse primers (highlighted in yellow) and the EcoRV restriction enzyme site (highlighted in magenta).(c) Colony morphologies of P. variotii isogenic lines on PDA.

Fig. 7 .
Fig. 7. Antagonistic potential of P. variotii isogenic lines.(a) Dual-culture assay and (b) percentage inhibition of P. variotii virus-infected (VI) and virus-free (VF) isogenic lines against three plant pathogenic fungi, A. terreus, Hypoxylon spp.and P. citrinum.The blue and red arrows in (a) indicate the colony diameter of the fungal pathogens in control (A) and treated (B) groups, respectively, as measured during exemplar dual-culture assays to calculate percentage inhibition in (b).Two-way ANOVA indicated an overall statistical significance with P-value <0.01 when comparing P. variotii isogenic lines.